The E2F1 transcription factor is involved in many diverse functions, such as cell cycle progression, differentiation, DNA repair and apoptosis. There has been a lot of progress in the understanding of its role and regulation during DNA damage, including new regulators and posttranslational modification. One of its regulators, TopBP1, is also involved in checkpoint activation. However, it is not clear whether and how the functions of TopBP1 in E2F1 regulation and checkpoint activation are coordinated. We have now elucidated how these functions are switched via a PI(3)K/Akt-dependent mechanism. This regulatory mechanism often goes awry in cancer. We have developed a new strategy to rectify this abnormality in cancer. In this proposal, we aim to determine how E2F1 apoptosis is regulated by these events and test this new strategy to activate the E2F1-dependent apoptosis for cancer therapy. First, We will investigate a new mechanism of how TopBP1 switches its functions between the checkpoint activation and E2F1 regulation and how it is perturbed in cancer. Second, we will investigate how E2F1 is regulated by K63-linked ubiquitination. We will determine the E3 ligase(s) responsible for K63-linked E2F1 ubiquitination and investigate how a deubiquitinase regulates E2F1 activity. Lastly, we will develop new therapeutic strategies to activate E2F1-mediated apoptosis in cancer. Upon completion of this project, these new data will greatly advance our understanding of the molecular mechanism controlling both transcription and checkpoint, and how this basic mechanism is commonly de- regulated in cancer. Our new data will also provide extensive knowledge regarding how K63-linked ubiquitination regulates E2F1 activity through the critical regulators (E3 ligases and a deubiquitinase enzyme). Importantly, for the first time, we will be able to harness E2F1 activity for promoting apoptosis in cancer cells.